Disclosed are methods of extracting and purifying capsinoids (e.g., capsiate, dihydrocapsiate) from Capsicum species fruit involving drying (e.g., freeze drying) the fruit to produce dried fruit and grinding the dried fruit to produce powdered fruit, extracting the powdered fruit at least once with a non-polar organic solvent (e.g., pentane, hexane, heptane, iso-octane, cyclohexane) to produce an organic solvent extract, subjecting the organic solvent extract at least once to liquid/liquid partitioning (no drying involved) using a polar solvent (e.g., acetonitrile, methanol) to form a capsinoid enriched polar solvent partition, and optionally purifying the capsinoid enriched polar solvent partition (e.g., via HPLC using acetonitrile as a carrier) to yield capsinoids. The non-polar organic solvent is not miscible with the polar solvent.
Capsaicinoids (the pungency factor in hot peppers) are vanillylamide moieties with C9-C11 branched chain fatty acids. Capsaicin (FIG. 1) is widely used in the pharmaceutical industry as a result of its various biological properties, including its ability to increase metabolic activity.
A family of capsaicinoid-related compounds called capsinoids was reported in 1998 (Kobata, K., et al., J. Agri. Food Chem., 46(5): 1695-1697 (1998)). Principle among these is capsiate and dihydrocapsiate (FIG. 1) which was first isolated from the pepper (Capsicum annuum L.) cultivar CH-19 (Kobata et al., 1998). Capsinoids are similar in structure to capsaicinoids but possess an ester group in place of the amide moiety. As a result of this structural change, capsinoids are not pungent and they do not have the adverse side-effects typically associated with capsaicin: application of capsaicin to the skin and its ingestion often result in severe irritation. Naturally-occurring capsinoids include capsiate, dihydrocapsiate, and nordihydrocapsiate (Kobata, K., et al., J. Nat. Prod., 62(2): 335-336 (1999)).
The scientific literature suggests that capsinoids possess various forms of biological activity similar to those of capsaicin and its analogs. For example, reports indicate that capsinoids enhance adrenal catecholamine secretion, promote energy metabolism, suppress body fat accumulation, and increase endurance (Onuki, K., et al., J. Nut. Sci. and Vit., 47(4): 295-298 (2001a); Onuki, K., et al., Bio., Biotech., and Biochem., 65(9): 2033-2036 (2001b); Onuki, K., et al., Bio., Biotech., and Biochem., 65(12):pp. 2375-2 (2001c); Haramizu S., et al., BioSci. Biotechnol. Biochem., 70(4): 774-781 (2006); Faraut, B., et al., Intnl. J. Obesity, 33:1348-1355 (2009)). Capsiate improves glucose metabolism by improving insulin sensitivity (Kwon, D. W. Y., et al., J. Nutr. Biochem., 24: 1078-1085 (2013)). Capsiate has been shown to be more effective than capsaicin in delivering the analgesic drug N-ethyl-lidocaine (QX-314). The use of capsaicin to transport QX-314 across membranes results in significant pain, whereas capsiate is equally as effective but causes no pain (Nakagawa, H., and A. Hiura, Austin Biomark Diagn., 1:4-9 (2014)). Like capsaicinoids, capsinoids possess significant antioxidant activity (Rosa, A., et al., J. Agri. Food Chem., 50: 7396-7401 (2002)).
We have developed a new method of effectively isolating capsinoids such as capsiate and its analogs from fruit of Capsicum species such as C. annum L.